The present disclosure relates generally to aminated materials for Enzyme-Linked Immunosorbent Assays (ELISAS) and processes, compositions and devices for improving the performance of such assays. Enzyme-Linked Immunosorbent Assay (ELISA) is a commonly used immunoassay. It has been widely used for detection and quantification of biological agents (mainly proteins and polypeptides) in the biotechnology industry, and is becoming increasingly important in clinical, food safety, and environmental applications. ELISA uses an enzymatic reaction to convert substrates into products having a detectable signal (e.g., fluorescence). Each enzyme in the conjugate can covert hundreds of substrates into products, thereby amplifying the detectable signal and enhancing the sensitivity of the assay.
The general principles and procedures used in ELISA are described here with reference to a 96-well microtiter plate. For example, the following procedures can be utilized:                (a) The first antibody (specific for the antigen to be assayed) is added to an ELISA plate. The first antibody is allowed to adsorb to the solid substrate surface. The excess antibody is removed from the plate by washing after incubation.        (b) The wells are filled with blocking solution. The blocking solution provides proteins, which adsorb to all protein-binding sites and prevent subsequent nonspecific binding of antibody to the plate.        (c) The sample is added. If the sample contains the targeted antigen, it will bond to the adsorbed first antibody to form an antigen-antibody complex. After incubation, the plate is washed.        (d) The conjugate solution is added. The conjugate (the second antibody) is an appropriate enzyme-labeled ligand (usually an antibody), which will bond to the antigen. The conjugate solution is discarded and the plate is washed after incubation.        (e) The developing solution containing the substrate is added, which reacts with the enzyme in the conjugate. Each enzyme is able to convert hundreds of substrate into products to enhance the sensitivity of the assay. The products of the reaction emit fluorescence or change the color of the solution.        
Conventional ELISA is typically carried out in 96-well microtiter plates. This involves a series of mixing, reaction, and washing steps, which not only are laborious but also often lead to large errors and inconsistent results. It usually takes several hours or longer to complete one assay because of the long incubation time required in each step. The long incubation time is a result of inefficient mass transport by molecular diffusion from the solution to the solid surface, although the immuno-reaction itself is a rapid process.
Microchip-based immunoassays have attracted attention for their potential advantages of having a high specific surface area, very low reagent consumption, and reduced assay time due to the device's microscale. Several microfluidic devices for immunoassays and enzyme assays have been developed and tested. For example, Lai and coworkers have demonstrated the feasibility of performing ELISA using a poly(methyl methacrylate) (PMMA) compact disk microfluidic platform as described in U.S. patent application Ser. No. 11/561,149, filed Nov. 17, 2006. However, because the microfluidic devices have a large surface-to-volume ratio, controlling their surface properties is a critical issue. In general, the sensitivity of the microfluidic device is dependent on the total activity of the antibodies or enzymes attached on the surface. How the surface interacts with antibodies and other biomolecules also affects the specificity of the immunoassay. Since the total surface area of a microfluidic device is fixed, it is important to attain a high immobilization activity yield during the immunoassay. The conventional passive adsorption of antibodies onto the surface is mainly driven by hydrophobic interactions, which often cause protein denaturation and reduce the protein's functional sites or activity by more than 90%. This problem increases when the microdevice has a large specific surface area. Developing an efficient surface modification method to enhance the binding efficiency and activity of the target protein is therefore important.
Polymers are low-cost alternative substrates materials for microfluidic devices, offering a wide range of physical and chemical properties that afford good processability for mass production and recyclability. However, efficient surface treatment methods for facilitating protein binding on the polymer surface are not well-developed. Several protein-polymer surface binding methods have been used in immunoassays, ranging from simple protein passive adsorption on polystyrene beads, adsorption via lipid layers grafted on poly(dimethylsiloxane) (PDMS), to adsorption via protein A bound to a PDMS surface. In developing fluorescence-based biosensors, PMMA has many advantages because it is transparent, has a low fluorescence background, and can be easily fabricated. However, direct adsorption of antibodies to the PMMA surface, without any surface modification, yields a low binding efficiency because the antibody binds poorly onto the untreated PMMA surface. Although various surface modification methods have been developed to introduce functional amine groups on the PMMA surface, they either consist of many steps and yield a low surface amine density, or involve unstable intermediates and environmentally unfriendly solvents in their preparation.
It would be desirable to provide a surface modification method which introduces functional amine groups onto a polymeric surface with few steps and yielding a high surface amine density.